Project Summary Complications from infections caused by difficult to treat Gram-negative pathogens resistant to multiple antibiotics (the MDR phenotype) has become a major public health threat. The basis of resistance in the most important members of this group, including Pseudomonas aeruginosa, is the poor penetration of the antibiotics through a relatively impermeable bacterial cell envelope and their expulsion (efflux) by various pumps expressed by the microorganisms. Consequently, the intracellular concentrations of antibiotic are kept below bactericidal levels. This proposal aims at creating new classes of diagnostic tools and therapeutic agents to combat efflux-based resistance by a project organized into two phases. First, we will develop genetically encoded fluorescent sensors activated by binding of antibiotics following their entry into the bacterial cytoplasm. This tool will enable us to measure antibiotic flux under different environmental conditions and on a single cell basis. During the second phase of the project, working with our industrial partner, we will implement a screening program, using a library of several hundred billion small molecules, to identify inhibitors targeting different components of efflux pumps. These inhibitors should activate the fluorescent detector by causing enhanced intracellular accumulation of the antibiotics resulting from inhibition of efflux. Functional studies are proposed to assess the ability of the active compounds to promote killing of P. aeruginosa by antibiotics that are normally expelled by the efflux pumps. Crystal structures of inhibitors bound to target proteins will be determined and these will guide medicinal chemistry efforts with the goal of creating additional derivatives with increased potencies and expanded spectrum, capable of inhibiting efflux pumps of other important Gram-negative pathogens.